Asian Journal of Mycology 2(1): 101–109 (2019) ISSN 2651-1339 www.asianjournalofmycology.org Article Doi 10.5943/ajom/2/1/4 A new species of Pseudocercospora on Encephalartos barteri from Benin Meswaet Y1, Mangelsdorff R1, Yorou NS2 and Piepenbring M1 1Institute of Ecology, Evolution and Diversity, Faculty of Biosciences, Goethe University Frankfurt am Main, Biologicum, Max-von-Laue-Str. 13, 60439 Frankfurt am Main, Germany 2Faculty of Agronomy, University of Parakou, BP 123 Parakou, Benin Meswaet Y, Mangelsdorff R, Yorou NS, Piepenbring M 2019 – A new species of Pseudocercospora on Encephalartos barteri from Benin. Asian Journal of Mycology 2(1), 101– 109, Doi 10.5943/ajom/2/1/4 Abstract An infection of leaves of Encephalartos barteri (Zamiaceae) by a cercosporoid fungus was repeatedly observed in central Benin, West Africa. Morphological characteristics, the host relationship and DNA sequence data for two gene regions, namely ITS and rpb2, were compared to the corresponding characteristics of closely related, known cercosporoid species and showed that the specimens from Benin represent a new species of Pseudocercospora. Pseudocercospora encephalarti is the first Pseudocercospora species on a species of the host genus Encephalartos, as well as for the whole class Cycadopsida. It was found to be closely associated with a species of Corynespora that could not be identified in the context of the present study. Key words – Corynespora – Cycadopsida – Mycosphaerellaceae – new species – Zamiaceae Introduction The genus Pseudocercospora was established by Spegazzini (1910) based on the type species Pseudocercospora vitis (Lév.) Speg., a foliar pathogen of grapevines. The majority of Pseudocercospora species known to date are pathogens on a wide variety of plants, including numerous economically relevant species of food crops or ornamentals all over the world (Den Breeÿen et al. 2006). They are known mainly from tropical and sub-tropical environments where they cause leaf spots, blight, fruit spots or fruit rot (von Arx 1983, Chupp 1954, Deighton 1976, Pons & Sutton 1988). Pseudocercospora species are morphologically characterized by pigmented conidia and conidiophores without thickened scars (Pereira & Barreto 2006). Species of this genus usually infest angiosperms, but a few species are also known from gymnosperms (Braun et al. 2013). Materials & Methods Collections and morphological studies Leaf samples infected by cercosporoid fungi were collected in Benin in July and August of 2016 and 2107. Specimens were observed by stereomicroscopy and by standard methods of light microscopy, using a Zeiss Axioscope 40 microscope. For light microscopy, leaf sections were made Submitted 14 January 2019, Accepted 11 April 2019, Published 25 April 2019 Corresponding Author: Yalemwork Meswaet – e-mail – [email protected] 101 with razor blades and mounted in distilled water and 5% KOH without any staining. Semi- permanent preparations of sections of the infected leaf were made by a microtome (Leica CM 1510-1) and mounted in lactophenol with cotton blue. Measurements of 30 conidia, conidiophores and other structures have been made at a magnification of ×1000. Measurements are presented as mean value ± standard deviation with extreme values in parentheses. For scanning electron microscopy, dried material was directly mounted and sputtered with gold for 3 minutes. Photographs were made with a Hitachi S 4500 scanning electron microscope (SEM). DNA Extraction and PCR amplification DNA was isolated from caespituli taken from dry specimens of the cercosporoid fungus using E.Z.N.A® Forensic DNA Extraction Kit following the manufacturer’s instructions with a few modifications. Small pieces of leaves containing several clean caespituli, with as little other fungi as possible, were checked under the stereomicroscope. Precautions were taken to avoid picking any other associated materials that could lead to potential contamination. To extract total genomic DNA from caespituli, a small amount of clean mycelium from the leaf surface was transferred into a sterile Eppendorf tube using a sterilized needle and tape-lifts. The material was homogenized for 7– 10 min. using a Retsch Mixer Mill MM301 with TL buffer and 2.5 mm Zirconia beads. Isolated DNA was re-suspended in elution buffer and stored at -20°C. DNA concentration was checked by a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA). Two genomic loci were amplified. For the ITS region, primers V9G (de Hoog & Gerrits van den Ende 1998) with ITS4 (White et al. 1990) and for the rpb2-locus Rpb2-F4 (Videira et al. 2017) or RPB2-5F2 (Sung et al. 2007) with fRPB2-7cR (Liu et al. 1999) were used. PCR amplification and sequencing were conducted following the protocols of Crous et al. (2009, 2012) and Videira et al. (2017). The PCR mixtures consisted of 1 μL genomic DNA, 15× MgCl2 reaction buffer (Bioline, Luckenwalde, Germany), 25 mM MgCl2, 25 μM of each dNTP, 10 μM of each primer and 5 U Taq DNA polymerase (VWR) in a total volume of 25 μL. Cycling parameters of the PCR for ITS were as follows: initial denaturation 94°C for 3 min; 35 cycles of amplification [denaturation at 94°C for 30 s, primer annealing 52°C for 30 s and TAQ extension 72°C for 45 s], and a final TAQ extension 72°C for 5 min, followed by storage at 8°C. The PCR mixture for rpb2 contained 2 μL of template DNA and to obtain the partial rpb2, a touchdown PCR protocol was used as described by Videira et al. (2017). PCR-products were checked on 1.5 % agarose electrophoresis gels containing HDGreenPlus DNA stain. Amplified PCR products were purified with the Cycle Pure Kit (VWR-Omega, USA). Sequencing was performed at Seqlab GmbH, Germany. Molecular phylogeny Amplification of the ITS and rpb2 gene regions for all isolates used in this study resulted in amplification products of approximately 650 bp for ITS and 1068 bp for rpb2. Consensus sequences of trace files were generated with Geneious 10.2.2 (https://www.geneious.com, Kearse et al. 2012) and searched against GenBank (https://www.ncbi.nlm.nih.gov/, Benson et al. 2013) with MegaBLAST. Sequences with a maximum identity of more than 95% (14 sequences to ITS and rpb2) were retrieved (Table 1). The sequences obtained from GenBank (Table 1) and sequences generated in this study were aligned with MAFFT v. 7 using the L-INS-i algorithm, (Nakamura et al. 2018). The alignments were manually checked by using MEGA v. 7 (Kumar et al. 2016). Gblocks v. 0.91b (Talavera & Castresana 2007) was used to remove poorly aligned positions and divergent regions from the DNA alignment using the parameters for a less stringent selection. To test the level of congruence among the two loci (ITS & rpb2), the Congruence Among Distance Matrices test, CADM global of R package APE v.3.2 (Paradis et al. 2004, R Core Team 2017), was performed. The CADM results showed that the null hypothesis of complete incongruence among loci was rejected (W = 0.0.89; p < 0.01), thus allowing concatenation of the two loci. Subsequently a two locus concatenated alignment (ITS, rpb2) dataset using Geneious 10.2.2 for phylogenetic analyses was assembled. Passalora eucalypti (CBS 111318) served as outgroup taxon as proposed 102 by Crous et al. (2012). PartitionFinder2 XSEDE v.2.1.1 (Miller et al. 2010) was used to select the best-fit model of evolution (K80+I+G model to ITS, and K80+I model to rpb2) for each gene fragment separately for Bayesian and Maximum Likelihood (ML) analyses. The alignment and the tree were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S23598). Phylogenetic analyses of this study were conducted by applying Maximum Likelihood (ML) with RAxML-HPC2 on XSEDE v.8.2.10 (Miller et al. 2010) and Bayesian with MrBayes on XSEDE v.3.2.6 (Miller et al. 2010) methods in the CIPRES Science Gateway web portal. (http://www.phylo.org/sub_sections/portal/). For Maximum Likelihood phylogenies performed with RAxML 1000 rapid bootstrap inferences were executed. For Bayesian phylogenies, two parallel runs with eight chains of Metropolis-coupled Markov chain Monte Carlo iterations were performed with the heat parameter being set at 0.2. Analyses were run for 100 million generations, with trees sampled every 1000th generation until the average standard deviation of split frequencies reached 0.01 (stop value). The first 25 % of saved trees were discarded as the ‘burn-in’ phase. Posterior probabilities (PP) were determined from the remaining trees. Bayesian posterior probabilities (BPP) ≥ 94 % and Bootstrap values (BS) ≥ 70% were considered significant. Table 1 Sequences downloaded from GenBank (in alphabetical order) used in this study Species Host Sources GenBank Accession No. References ITS rpb2 Passalora eucalypti Eucalyptus CBS 111318 KF901613 KF902267 Quaedvlieg et al. saligna 2014 Pseudocercospora Breonadia CBS 143489 MH107913 MH108006 Crous et al. 2018 breonadiae salicina Pseudocercospora Terminalia MAFF MF951366 MF951616 Videira et al. 2017 catappae catappa 238312 Pseudocercospora Haloragis erecta CBS 114645 KX287299 KX288454 Videira et al. 2016 dingleyae Pseudocercospora Eucalyptus CBS 124990 GU269799 MF951619 Videira et al. 2016 flavomarginata camaldulensis Pseudocercospora Eucalyptus CBS 124155 KF901673 KF902318 Quaedvlieg et al. madagascariensis camaldulensis 2014 Pseudocercospora Nelumbo Kirschner KY304492 LC199940 Chen & Kirschner nelumbonicola nucifera 4111 2018 Pseudocercospora Nerium oleander CBS 138010 NR137885 KX462647 Nakashima et al. neriicola 2016 Pseudocercospora Prunus CBS 132107 GU269676